A significant bottleneck for translation of hESC derived cardiomyocytes to study adult-onset human cardiac diseases in vitro and for treatment of human myocardial infarction in vivo are their abilities to mature and/or survive. We have developed a method to direct hESC to the cardiac lineage, using a staged protocol that involves the formation of a primitive-streak-like population, the induction and specification of cardiac mesoderm and the expansion of the cardiovascular lineages, which can be exploited to study hESC differentiation. The extracellular matrix (ECM) could be a critical determinant of directing human cardiac stem cell differentiation because of its effects in promoting early mouse ESC-derived cardiomyocyte differentiation in vitro. However, there are limited studies, which have assessed the role of the myocardial matrix on directing human cardiac stem cell differentiation in vitro and in vivo. We recently developed a method to extract myocardial matrix from adult porcine cardiac muscle, which can be solublilized and absorbed on tissue culture dishes as a coating for in vitro cell-based studies and can be solubilized as an injectable biocompatible material in vivo. We show the maturation potential of this myocardial matrix by demonstrating that hESC derived cardiomyocytes displayed a significant increase in myofibrillar growth and cell-cell junction (desmosomal) maturation/ differentiation as opposed to cells plated on traditional gelatin substrate, which displayed a more punctate "fetal" expression pattern of desmosomal localization. The overall goal of this two-year proposal is to identify whether the myocardial matrix is an important determinant of hESC derived cardiomyocyte differentiation/maturation in vitro and in vivo. These results led us to the hypotheses that tissue-specific cues from the ECM are required to direct hESC-derived cardiac cell survival, maturation/differentiation and function in vitro and in vivo. We will achieve these goals by comprehensive molecular, cellular and functional analyses of the effects of the myocardial matrix on hESC-derived cardiomyocytes in vitro and in a rat model of myocardial infarction in vivo. Specific Aims include: (1) To determine whether the myocardial matrix is a cellular determinant of hESC derived cardiomyocyte maturation, differentiation and function in vitro. (2) To rescue/alter death and progression of myocardial-infarction induced heart failure in rats by injecting myocardial matrix in combination with hESC-derived cardiomyocytes into the infarcted myocardium in vivo. PUBLIC HEALTH RELEVANCE: We propose to develop new technologies to direct stem cell differentiation, thus enabling the more timely understanding of stem cell biology and the advancement of new stem cell based therapies for heart disease. Stem cell derived cardiomyocytes will be the initial focus;however, these technologies will be applicable to other stem cell fields.